Flavone‐associated resistance of two Lemna species to duckweed weevil attack

Abstract Lemna perpusilla and Lemna minor are free‐floating plants that often live in the same habitat. However, little is known about how they differ in response to herbivore attacks. In this study, we examined the species‐specific resistance of two Lemna species to the duckweed weevil, Tanysphyrus lemnae. The female adults of T. lemnae preferred to lay eggs on L. perpusilla over L. minor. In addition, the larvae of T. lemnae performed better when fed on L. perpusilla than on L. minor. To understand the physiological basis of species‐specific resistance in the two Lemna species, we measured the amounts of jasmonic acid (JA), phytosterols, and flavonoids. Attacks by duckweed weevils increased the levels of JA in the two Lemna species, but these levels did not differ significantly between the two species. Interestingly, the levels of flavones (isoorientin, vitexin, and isovitexin) in L. minor species were higher than those in L. perpusilla. The in vitro bioassay showed that three flavones significantly decreased the survival rate of duckweed weevil larvae. Although L. perpusilla was less resistant to duckweed weevil attack compared to L. minor, L. perpusilla grew faster than L. minor regardless of the duckweed weevil attack. These results suggest that these two Lemna species have different defense strategies against the duckweed weevil.


Plants interact with herbivorous insects in various ecosystems.
Most studies have involved terrestrial plants and herbivores because interactions in terrestrial ecosystems are regarded as more diverse and abundant than those in aquatic ecosystems (Forister et al., 2015;Hay & Fenical, 1988;Vermeij, 2016). Although the interaction between aqu atic plants and herbivorous insects is poorly understood (Vermeij, 2016), herbivorous insects are known to negatively affect plant abundance in aquatic habitats (Bakker et al., 2016;Wood et al., 2017). For instance, herbivorous insects in aquatic environments reduce plant growth and viability (Doyle et al., 2007;Owens et al., 2006). In addition, aquatic herbivores affect aquatic restoration, plant reproduction, and plant competition in aquatic environments (Harms et al., 2011;Van et al., 1998). Therefore, it is necessary to understand the interaction between aquatic plants and herbivorous insects at the community level.
Aquatic herbivorous insects integrate many factors when selecting a host plant for oviposition and offspring feeding (Cherian et al., 2001;Scheirs & Bruyn, 2000;Thompson, 1988); the levels of primary as well as secondary metabolites are key factors for host selection (Anderson et al., 2011;Awmack & Leather, 2002).
For instance, the phenolic secondary metabolites of aquatic plants were negatively correlated with herbivore feeding preferences (Lodge, 1991;Vergeer & Van Der Velde, 1997). Flavonoids play various roles in plants. Flavonoids regulate plant growth and photosynthesis, and they protect cells from UV-damage, drought, and pathogen and herbivore attacks (Pagliuso et al., 2020;Pichersky & Gang, 2000;Yonekura-Sakakibara et al., 2019). The defense-related flavonoids can be divided into two categories: the inducible flavonoids, which are induced by abiotic and biotic stresses, and the constitutive flavonoids, which are synthesized during normal development (Treutter, 2005). The constitutive metabolites are stored in strategically important tissues, such as flowers and fruits (Brunetti et al., 2013;Taylor & Grotewold, 2005). Some flavonoid metabolites of the aquatic fern Azolla pinnata act as chemical deterrents to aquatic herbivores (Cohen et al., 2002). In addition, submerged Elodea plants contain flavone glucosides, which reduce the feeding preference and growth of aquatic herbivorous Lepidoptera (Erhard et al., 2007). In the case of phytohormones, several studies mentioned the possible role of phytohormones in Lemnaceae family. For instance, exogenously applied JA (jasmonic acid) and ABA (abscisic acid) at low concentrations were found to induce flowering in Spirodela polyrrhiza and Lemna minor (Krajncic et al., 2006;Piotrowska et al., 2010). Another study has tested the effect of exogenous phytohormones (SA; salicylic acid, ABA and JA so on) in the growth regulation of L. minor (Utami et al., 2018). Most studies focused on the effect of the exogenous application of phytohormones but not the endogenous function of phytohormone in Lemna species.
In nature, plants have to cope with various biotic stresses and constantly face attacks by pathogens and herbivorous insects (Baldwin, 2001;Walling, 2009). In addition, plants need to balance growth and defense to optimize their fitness (Huot et al., 2014).
Growth-defense tradeoffs drive differential intra-or inter-species competition and affect the susceptibility of host plants to herbivore attack (de Vries et al., 2017). Because many aquatic plants grow fast, aquatic plants can be a useful model system for studying growthbiased strategies in the growth-defense trade-off phenomenon in plants (Acosta et al., 2021).
Lemnaceae are the smallest and fastest-growing flowering plants (Kurepa et al., 2018;Laird & Barks, 2018). Duckweeds have a leaflike structure called a frond (or thallus), no stem, and one or more roots. Although duckweeds can produce flowers, they normally propagate vegetatively (Hillman, 1961). Duckweeds comprise five genera (Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia) and 36 species in the world (Acosta et al., 2021). Species of the common duckweed, Lemna, are distributed globally (Silva et al., 2018). Among the genus Lemna, L. minor has been extensively studied, because L. minor plants are also used for phytoremediation and dietary supplements (Wang et al., 2016). Moreover, small genome sizes, genetic manipulation techniques that enable functional testing of genes, and rapid vegetative propagation make it suitable for a variety of research applications including biochemistry, metabolism, and interactions with microbial communities (Acosta et al., 2021). The preferences of water-lily aphid preferences for four duckweed species were examined recently, but it has not been tested mechanism of preference of the aphid (Subramanian & Turcotte, 2020). On the other hand, most studies on the chemical side of Lemna species focused on metabolic changes (e.g., starch, phenolics and flavonoids) in responses to abiotic stress (Pagliuso et al., 2020;Tao et al., 2017).
Despite their central roles in aquatic ecosystems and their heavy use in biotechnology, the defense responses of Lemna species to herbivorous insects are still poorly understood.
The purpose of this study was to examine how two aquatic free-floating plants, L. perpusilla and L. minor, defend against a duckweed weevil, Tanysphyrus lemnae, attack. We hypothesized that two Lemna species have different chemical defenses against herbivores.
In order to test this hypothesis, we first examined the duckweed weevil's oviposition preference and larval performance in two Lemna species. Phytohormones and metabolites were then measured in two Lemna species in response to weevil attacks. Lastly, we examined the defensive role of metabolites and the growth-defense trade-off in both Lemna species.

| Plant and insect materials and growth conditions
Wild-type L. perpusilla (minute duckweed) was collected from a natural population in Daejeon, South Korea. L. minor (common duckweed) was obtained from the biological resource center of the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Jeonbuk, South Korea), and was originally collected from Jeju island, South Korea. We propagated L. perpusilla and L. minor plants from a single colony. Sterilized colonies were grown in fertilizer solution (Kinnersley & Lin, 2000) for experiments and insect colony maintenance.
Lemna perpusilla and L. minor are distributed in the same habitats, such as wetlands, slow-flowing streams, upper estuaries, paddy fields, agricultural waterways, lakes, and ponds, in South Korea. During maturation, the daughter fronds of Lemna species are launched from the axial meristematic zone of the mother frond.
The central position of the daughter-mother frond is connected by a stipule; this stipule breaks off after maturation (Cherian et al., 2001;Topp et al., 2011). To differentiate between the two sibling species, we confirmed that the frond of L. perpusilla has a winged root sheath at its base, an ovate shape, thalli without anthocyanin pigment, and a lighter green leaf color than the frond of L. minor (Hillman, 1961;Landolt, 1986) (Figure 1a). All plants were grown at 25 ± 2°C, 16 h light/ 8 h dark cycle with 60% relative humidity and 100 μmol m −2 s −1 of white light in the growth room.
Tanysphyrus lemnae is a weevil of aquatic herbivorous insect distributed native to Europe to North America and occurs through Asia and Japan. Tanysphyrus lemnae refers to the host plant, Lemna genera (Paykull, 1792). Adult weevils chew on the fronds using chewing mouthparts located at the end of long snouts, creating round holes.
The larvae of T. lemnae also feed on the fronds, but the larvae tunnel through the frond in curved patterns like miners. We collected adult duckweed weevils of T. lemnae from the same pond in which L. perpusilla plants were collected (Figure 1a). We developed T. lemane weevil colony in laboratory conditions for further experiments. The adults were reared in a plastic cage with a ventilation hole that was covered with a nylon mesh (diameter 12 × height 8 cm, hole 4 cm, insect breeding dish, SPL, South Korea) and allowed to mating and ovipositing. Tanysphyrus lemnae females laid one egg on the fronds' abaxial (lower) part per one frond. The fronds with an oviposited egg were collected and placed in a new cage. One week after moving (ovipositing), the eggs hatched and the larvae began feeding on the intact frond for a week. After that the larvae became pupation stage.
Newly adults hatched five to seven days later, (Figure 1b). Insects were maintained under the same conditions as duckweed.

| Adult preference and larval performance
Adult preference was estimated by measuring the feeding area and by counting eggs in plastic cages same as the insect colony cage (diameter 12 × height 8 cm, SPL, South Korea) that contained two compartments. We placed the same area of the two Lemna species, L. perpusilla and L. minor to eliminate feeding and oviposition biases caused by differences in frond areas (10 L. perpusilla trifoliate fronds and 15 L. minor trifoliate fronds). One gravid female weevil was placed in the middle of each cage. For the preference test, we placed the two Lemna species in each custom-made small plastic plate (diameter 40 mm x height 8 cm) with the central part removed for easier floating. Eighteen cages were used for the preference assay.
The two Lemna species were randomly placed to avoid positional effects. We first allowed newly born females to mate for five days, and then released the mated females for three days. After that, we measured the area of fronds that had been consumed by the female feeding using ImageJ software, and we counted the number of eggs oviposited on fronds using a microscope (SMZ645, Nikon).
To examine the larval performance of T. lemnae species, we measured the length of larvae feeding on each species of L. perpusilla and L. minor species. Each 500 fronds of L. perpusilla and L. minor species were placed in each plastic cage (L 21 × W 21 × H 6 cm, ventilation hole diameter 10 cm with 300 μm aperture mesh, BugDorm, MeagView Science Co.) for a sufficient amount of food was supplied during the experiment period. Duckweed weevils (25 males and 25 females) were released for mating and ovipositing in the cage, then removed after 1 day. After 7 days, we collected larvae. To quantify the length of larvae, 10 larvae were collected each from L. perpusilla and L. minor and took pictures to process the images with ImageJ software (1.53 e, National Institutes of Health).

| Phytohormone analysis
Six-pooled frond samples were collected from each duckweed species, both intact and damaged by T. lemnae adults for an hour. There are six biological replicates. The method of phytohormone extraction (JA; jasmonic acid, SA; salicylic acid and ABA; abscisic acid) and quantification were previously described in Joo et al. (2021). Briefly, approximately 100 mg of each frozen frond sample was homogenized with two steel beads in a TissueLyser II (Qiagen) for 1 min at 26 Hz after adding 1 ml ethyl acetate spiked with internal standards mixture: 20 ng each of [ 2 H 2 ] JA, [ 2 H 4 ] SA and [ 2 H 6 ] ABA. The extracted samples were centrifuged at 16,100 g at 4°C for 20 min, and F I G U R E 1 Aquatic duckweed weevil, Tanysphyrus lemnae feeds on two Lemna species in a native habitat (a) T. lemnae adults, L. perpusilla, and L. minor were collected from their native habitat. (b) Developmental stages of the duckweed weevil, T. lemnae. The female deposits one egg (red arrow) per frond. After 5-7 days, an egg hatches (red arrow) inside a frond, and a larva starts to feed, moving freely to a new frond when needed. After about 2 weeks, the larva begins to pupate (red arrow) inside of the frond. An adult emerges after 7 days. the supernatant was transferred into another new tube. The samples were evaporated to near dryness in a centrifugal vacuum concentrator (VC2124, Gyrogen) at 30°C. The dried samples were dissolved in 500 μl 70% (v/v) methanol: water for analysis with ultra-high performance liquid chromatography (UHPLC) triple-quadrupole mass spectrometry (LC-MS-8050, Shimadzu) as described previously (Joo et al., 2021). The phytohormones were detected in negative electrospray ionization mode (ESI), and the detailed detection method followed by Schäfer et al. (2016). The amounts of phytohormones were normalized by dividing the peak area of each phytohormone by the exact fresh mass of plant materials and the internal standards of each phytohormone.

| Phytosterol and primary metabolite analysis
We followed the extraction procedure described by Suh et al. (2013) for the phytosterols (campesterol, stigmasterol, and beta-sitosterol) and primary metabolite analysis. There are six biological replicates.
In each of the two Lemna samples, 10 mg was freeze-dried and ex-

| Flavonoid analysis
To examine flavonoid compounds in the two Lemna species, two Lemna species were released into each plastic cage that was used for the insect colony maintenance. We allowed 15 adult duckweed weevils to feed on plants for 10 days. There were five biological replicates. Approximately 100 mg of frozen materials was homogenized with a steel pestle and extracted by adding 200 μl of the extraction buffer (75% methanol/ 0.1% formic acid) as described in (Gomez et al., 2018). Supernatants were collected after ultrasound treatment for 30 min followed by centrifugation at 16,000 g at 4°C for 30 min.
The procedures were repeated twice. The collected supernatants were lyophilized for 24 h and resuspended in distilled water. The samples were stored in the freezer at −80°C until analysis. Aliquots of 300 μl of the re-suspended samples were transferred into amber vials with an insert were analyzed by UPLC-MS/MS (LC-MS-8050, Shimadzu), and 1 μl of the extracts was injected by the autosampler into the LC-MS system and chromatographic separation were carried out on a C18 column (UPLC BEH, 1.7 μm particle size, 100 mm length × 2.1 mm inner diameter, Waters). The solvents used in the mobile phases were deionized water containing 0.02% acetic acid (A); solvent B consisted of 0.02% acetic acid in acetonitrile, with the following concentration gradient of B: 5%, 0 min; 60%, 11 min; 95%, 13 min; 95%, 15 min; 5%, 16 min; 5%, 17 min. The mass spectrometer was operated in positive ESI mode using the same chromatographic conditions. Using Q3 scan and MRM (multiple reaction monitoring) methods, we analyzed a total of 18 target flavonoid compounds: four flavonols, six flavones, four flavanones, two chalcones, and two isoflavones (Table S1). All standard compounds were obtained from ChemFaces (Wuhan ChemFaces Biochemical Co.). Flavonoid compounds (isoorientin, vitexin, isovitexin, hesperetin, luteolin, and apigenin) were quantified by comparing their peak areas with calibration curves at a concentration of from 0.1 to 100 μg/ml.

| In vitro bioassay of duckweed weevil larvae
In vitro bioassays were conducted using semi-artificial diets sup-

| Growth performance of Lemna species
To examine the growth difference between two Lemna species, we Institutes of Health), and the damaged area by the T. lemnae weevil was excluded from the quantification.

| Statistical analysis
The consumed frond area and the number of eggs deposited on each pair of the two Lemna species for use as choice assay and the quantified value of larvae size were analyzed by a student's t-test.
Metabolite contents were analyzed by two-way ANOVA followed by Tukey's honestly significant difference (HSD) as post hoc test, and larval survival curves of treatment and control samples were compared by the log-rank test. The total frond area to see growth differences between the two Lemna species was analyzed by repeated measures t-test. The frequency distribution of larval preference were compared by G-test. The primary metabolites profiling of two

| Preference and performance of duckweed weevil, Tanysphyrus lemnae, that fed on two sibling Lemna species
To examine the preference of duckweed weevil for two sibling Lemna species, we conducted dual-choice assays that measured the insects' feeding and oviposition preferences. Tanysphyrus lemnae females consumed 2.65 times more fronds area of L. perpusilla than of L. minor (p < .05, Figure 2a). Tanysphyrus lemnae females also oviposited 1.74 times more eggs on fronds of L. perpusilla than of L. minor (p < .05, Figure 2b). To quantify larval performance, we measured the length of larvae fed on L. perpusilla and L. minor 7 days after oviposition (Figure 2c). The larvae fed on L. perpusilla were significantly bigger than those fed on L. minor. The size difference was 1.74 times more on the larval fed on L. perpusilla than L. minor (p < .05, Figure 2d).
We further investigated larval preference and found more larvae on fronds of L. perpusilla than on fronds of L. minor ( Figure S1).  (Figure 4).

| Species-specific defense responses of two sibling Lemna species to attack by duckweed weevil
In addition, we analyzed phytosterols -campesterol, stigmasterol, and β-sitosterol -in two Lemna species. Although none of the three phytosterols were induced by herbivore attack, campesterol and β-sitosterol accumulated in a species-specific manner ( Figure 4g-i, p < .05). We also conducted clustering analyses of primary metabolites in the Lemna species damaged by duckweed weevil on both sibling Lemna species. The heatmap analysis indicated that some primary metabolites were highly induced, especially in the attacked L. minor ( Figure S3a). Principal component analysis (PCA) showed a differential grouping for the two Lemna species ( Figure S3b).

| Lemna minor-specific flavones decrease larval survival of duckweed weevil
Because L. minor resistant than L. perpusilla to the duckweed weevil, we hypothesized that specific flavones in L. minor increased its resistance to T. lemnae. To evaluate the defensive roles of isoorientin, vitexin, and isovitexin, which accumulated mainly in L. minor ( Figure 4), we fed the early stage of T. lemnae larvae on semi-artificial diets spiked with each of these compounds. Because L. perpusilla barely produced any amount of these, we mixed freeze-dried L. perpusilla powder with agar to make the diet. Analysis of larval survival using the Kaplan-Meier method indicated that the level of all three L. minor-specific flavones significantly decreased the survival rate of duckweed larvae (log-rank test, Figure 5).

| The growth of the sibling Lemna species differed in response to herbivore attack
Next, we compared the frond area of two Lemna species with or without T. lemnae treatment. We placed 30 trifoliate fronds of L.
perpusilla and 48 trifoliate fronds of L. minor in each plastic cage (Day 0) and measured the total area of fronds three times at twoday intervals for 6 days. Significant difference was found in the total area of fronds between L. perpusiila and L. minor without herbivore attacks 6 days after release; L. perpusilla fronds grew faster than L. minor fronds (p < .05, Figure 6a). Fronds of L. perpusilla grew faster than those of L. minor between 2 and 4 days against duckweed weevil attacks (p < .01 and p < .05, Figure 6b). The value of frond area of L. perpusilla under duckweed weevil attack had higher than the frond area of L. perpusilla under control as well at 2 days after treatment (Figure 6a,b). Six days after plants were placed in the cage, the fronds of both Lemna species were fully expanded, and no difference was found.

| DISCUSS ION
In the preference and performance assays, we found that female duckweed weevils of T. lemnae preferred to oviposit and feed on L. perpusilla over L. minor (Figures 2a,b). The larvae on L. perpusilla grew larger than those on L. minor (Figures 2c,d). These results suggest that females of the aquatic herbivorous insect T. lemnae select host plants to optimize offspring performance, which supports the F I G U R E 3 Mean (±SE) levels of jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA) in Lemna species when damaged by T. lemnae adults. Asterisks indicate significant differences between species and treatments (**p < .01; ***p < .001, ns, no significant, n = 6) as determined by two-way ANOVA analysis. Different letters indicate significant differences between two Lemna fronds determined by ANOVAs with post hoc tests with Tukey correction (con; control, damaged; damaged by the duckweed weevil, T. lemnae).
optimal oviposition theory; T. lemnae are able to choose suitable plants for their offspring (Akol et al., 2013;Lee et al., 2016;Zhang et al., 2012). Further experiments are needed to identify oviposition factors of the duckweed weevil.
Among plant defense hormones, JA plays an important role in the regulation of plant defense responses to attacks by herbivorous insects (Erb et al., 2012;Pieterse et al., 2012). Although exogenous treatment of JA increase rice resistance against rice water weevil attack (Hamm et al., 2010), it has not been tested whether endogenous JA levels of aquatic herbivore are also induced by aquatic herbivore attack. Our results show that the levels of JA was highly induced in the fronds of both Lemna species, L. perpusilla and L. minor by duckweed weevil, T. lemnae attacks (Figure 3a), which support the hypothesis that the defensive responses of free-floating Lemna species to herbivorous insects is JA dependent as known in terrestrial plants. In both control and damaged L. minor, the levels of ABA were significantly higher than those in L. perpusilla. The results suggest that L. minor may be more sensitive to abiotic stress response than L. perpusilla, other than herbivore-induced stress. It is known that ABA plays a crucial role under various environmental stress conditions including cold, drought, and salt (Erb et al., 2012). Considering both species grow primarily in summer on the water, there is a strategy to adapt to salinity conditions that may change seasonally in nature (Marcos et al., 2018). However, the levels of major flavonoids in the two Lemna species were not induced by duckweed weevil attack but showed significant differences in contents between two sibling Lemna species. Although our results suggest that Lemna accumulates defensive metabolites at high levels even without herbivore attack (Erhard et al., 2007), other toxic secondary metabolites may also be induced by JA when herbivores attack.
we need to collect more genotypes of each species. Unique defensive mechanisms might be developed in L. minor and L. perpusilla under harsh or specific environmental conditions (e.g., interspecific competition among duckweeds) (Hart et al., 2019).
The flavonoid compositions of several duckweed species has been studied, because they have value in human health, medicine, and bioenergy (Böttner et al., 2021;Pagliuso et al., 2020;Tao et al., 2017;Wang et al., 2014). For instance, a giant duckweed, S. polyrhiza, contains four major flavonoids; cynaroside, orientin, apigetrin, and vitexin and the levels of those flavonoids vary in response to abiotic factors (Böttner et al., 2021). In addition, various flavanol glucoside and cycloartane glucoside have been investigated in Landoltia punctate (Wang et al., 2014). Another Lemna species, L. gibba has been indentified three major flavonoids, luteolin glucosides and vitexin, and this previous study showed that five genera of Lemnaceae identified different flavonoid profiling with significant contents of apigenin and luteolin derivatives (Pagliuso et al., 2020).
Consistent with other duckweed studies, our results showed that the dominant flavones -isoorientin (luteolin derivative), vitexin, and isovitexin (apigenin derivatives) -are detected constitutively in L. minor. However, little is known about defense functions of the flavonoid compounds in duckweed species against aquatic herbivore attacks. In this study, we showed that the three major flavones protect the fronds against aquatic herbivore weevil attacks ( Figure 5).
In case of other macrophyte species, the submerged macrophyte Elodea nuttalli plant produced has been studied for functional test of some flavonoid compounds such as luteolin, apigenin, and chrysoeriol-7-O-diglucuronide in response to against aquatic herbivorous Lepidoptera attacks (Erhard et al., 2007).
In this study, we show that two aquatic free-floating Lemna species make different growth-defense tradeoffs against herbivorous insect attacks. L. perpusilla grew faster than L. minor with or without herbivores (Figure 6b), while, L. minor accumulated more defensive substances that reduce the larval survival rates of T. lemnae (Figures 4 and 5). This result suggests that L. minor invests more energy in producing toxic metabolites rather than in promoting growth. Further studies are required to (a) show how growth-defense tradeoffs in the two Lemna species in response to aquatic herbivore attack are regulated at the molecular level, (b) examine the ecological consequences of plant resource allocation, and (c) elucidate the defensive role of two L. perpusilla-specific flavones: hesperetin and apigenin.

ACK N OWLED G EM ENT
We thank lab members in KAIST and CBNU for help with experiments and colony maintenance; Emily Wheeler for editorial assistance. This work is supported by the National Research Foundation of Korea (NRF-2019R1A6A3A01090595 andNRF-2018R1A5A7025409).

CO N FLI C T O F I NTE R E S T
No conflicts of interest or competing interests to report. F I G U R E 5 Survival curves of T. lemnae larvae fed on artificial diets spiked with different flavonoid compounds. Survival probability was estimated using the Kaplan-Meier method (rog-rank test, *p < .05; ***p < .001, n = 25).

F I G U R E 6
Growth difference of two sibling Lemna species. Mean frond area (±SE) of control and damaged Lemna species. An asterisk indicates significant differences (repeated t-test, *p < .05; **p < .01, n = 3).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data for the study are deposited in Dryad: https://doi. org/10.5061/dryad.stqjq 2c6h.